Amelioration of the development of cataracts and other ophthalmic diseases

ABSTRACT

Ophthalmically acceptable compositions used in arresting the development of cataracts or macular degeneration comprising a pharmaceutically acceptable carrier or diluent and a compound having the formula: 
     
       
         
         
             
             
         
       
         
         where R 1  and R 2  are, independently, H or C 1  to C 3  alkyl; 
         R 3  and R 4  are, independently C 1  to C 3  alkyl; and 
         where R 1  and R 2 , taken together, or R 3  and R 4 , taken together, or both may be cycloalkyl; 
         R 5  is H, OH, or C 1  to C 6  alkyl; 
         R 6  is or C 1  to C 6  alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl; 
         R 7  is C 1  to C 6  alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl 
         or where R 6  and R 7 , or R 5 , R 6  and R 7 , taken together, form a carbocycle or heterocycle having from 3 to 7 atoms in the ring.

This application claims benefit of Provisional Application No.60/381,287, filed May 17, 2002, the entirety of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention is directed to compositions that ameliorate thedevelopment of cataracts in the eye of a patient and to methods foreffecting such amelioration. In preferred embodiments of the invention,cataract development or growth is essentially halted. The presentinvention is also directed to the treatment of macular degeneration inthe eye and to certain other uses. In accordance with preferredembodiments, the compositions of this invention are capable ofadministration to patients without the need for injections and can beformulated into eye drops for such administration. Methods for treatmentof cataracts and macular degeneration are also provided, as are methodsfor the preparation of the novel compounds and compositions useful inthe practice of the invention.

BACKGROUND OF THE INVENTION

Aging-related cataract results from gradual opacification of thecrystalline lens of the eye. This disease is presently treated bysurgical removal and replacement of the affected lens. It is believedthat once begun, cataract development proceeds via one or more commonpathways that culminate in damage to lens fibers. This conditionprogresses slowly and occurs predominantly in the elderly.Alternatively, cataract may form because of surgical, radiation or drugtreatment of a patient, e.g. after surgery of an eye to repair retinaldamage (vitrectomy) or to reduce elevated intraocular pressure;x-irradiation of a tumor; or steroid drug treatment. A significantretardation of the rate of cataract development in such patients mayeliminate the need for many surgical cataract extractions. Thisreduction would provide tremendous benefits both to individual patientsand to the public health system.

It has been known to provide certain hydroxylamine compositions for theprevention or retardation of cataracts in the eyes of persons. U.S. Pat.No. 6,001,853, in the name of Zigler, et al., the content of which isincorporated herein by reference, reflects work performed at theNational Institutes of Health of the United States. Zigler et al.identified a class of hydroxylamines which, when administered to the eyeof a test animal, ameliorates cataract genesis or development. Suchadministration was necessarily via injection for physico-chemicalreasons. While Zigler stated in Example 6, it would be clinicallyconvenient to deliver TEMPOL-H by liquid eye drops, no working examplewas reported, Zigler's hydroxylamines being actually administered bysubconjunctival injections. Zigler's materials were also accompanied bythe co-administration of a reducing agent, either via injection,systemically or otherwise. It is believed that subsequent work at theNational Institutes of Health was directed to the identification ofeffective hydroxylamines that could be administered topically, howeverthose efforts were not successful.

Accordingly, it has been the object of intense research activity toidentify compounds and compositions containing them that can amelioratecataract formation and development in the eyes of patients without theneed for unpleasant, inconvenient and potentially dangerous intraocularinjections. In particular, a long-felt need has existed, which has notbeen fulfilled, for such compounds and compositions which can beadministered via topical application, especially via eye drops. Thisneed is addressed by the present invention.

Age-related macular degeneration is a leading cause of blindness in theUnited States and many European countries. The “dry” form of the diseaseis most common. It occurs when the central retina has become distorted,pigmented, or most commonly, thinned. The neovascular “wet” form of thedisease is responsible for most severe loss of vision. The wet form ofmacular degeneration is usually associated with aging, but otherdiseases which can cause wet macular degeneration include high myopia(being very nearsighted), some intraocular infections likehistoplasmosis, and AIDS. Accordingly there is a need for compositionsfor treatment of such ailments being easily deliverable to the eye ofpatients in great need.

SUMMARY OF THE INVENTION

The present invention provides compositions for the treatment ofcataracts in the eyes of patients either who are developing cataracts orwho are known or suspected of being at risk for formation of cataracts.Compositions are also provided for the treatment of macular degenerationin the eyes of patients who may exhibit or will likely exhibit maculardegeneration due to disease. In accordance with preferred embodiments,such compositions are formulated in topical liquid form, especially aseye drops. Periodic application of the compositions of this inventionretards or halts development of cataracts or macular degeneration intreated eyes. The invention provides compositions, which need not beapplied via injection or other uncomfortable or inconvenient routes.

In accordance with preferred embodiments, the present invention providescompositions comprising an ophthalmologically acceptable carrier ordiluent and a compound having the formula:

In such compounds, R₁ and R₂ are, independently, H or C₁ to C₃ alkyl andR₃ and R₄ are, independently C₁ to C₃ alkyl. It is also possible, inaccordance with certain embodiments, that R₁ and R₂, taken together, orR₃ and R₄, taken together, or both form a cycloalkyl moiety. In thecompounds of the invention, R₅ is H, OH, or C₁ to C₆ alkyl while R₆ isC₁ to C₆ alkyl, alkenyl, alkylyl, or substituted alkyl or alkenyl. R₇ isC₁ to C₆ alkyl, alkenyl, alkynyl, or substituted alkyl or alkenyl orC₁-C₆ cycloalkyl or heterocyclic. It is also possible for R₆ and R₇, orR₅, R₆ and R₇, taken together, to form a carbocycle or heterocyclehaving from 3 to 7 atoms in the ring. The term “ophthalmic,” as usedherein, means to have usefulness in the treatment of the eye and itsdiseases.

In the compounds used in the compositions of the invention, thesubstituted alkyl or alkenyl species can be substituted with at leastone hydroxy, alkoxy, alkylthio, alkylamino, dialkylamino, aryloxy,arylamino, benzyloxy, benzylamino or heterocyclic or YCO-Z where Y is O,N, or S and Z is alkyl, cycloalkyl or heterocyclic or aryl substituent.In accordance with some embodiments, the heterocycle is a 5, 6, or 7membered ring with at least one oxygen, sulfur, or nitrogen atom in thering. In one preferred composition, R₆ and R₇, taken together arecyclopropyl, while in others, R₆ and R₇, taken together aretetrahydrofuranyl and R₅, R₆ and R₇ taken together are furanyl.

For certain preferred compounds, each of R₁ through R₄ is C₁ to C₃alkyl, most especially ethyl or methyl, most especially, methyl. Forsome preferred embodiments, the compounds of the invention R₆ is C₁ toC₆ alkyl substituted with at least one C₁ to C₆ alkoxy or benzyloxygroup.

In other preferred compounds, each of R₁ through R₄ is methyl, R₅ is Hor methyl, R₆ is methyl substituted with benzyloxy or C₁ to C₆ alkoxyand R₇ is methyl or where R₆ and R₇ form a cyclopropyl group. In others,each of R₁ through R₄ is methyl, R₅ is methyl, R₆ is ethoxy methyl andR₇ is methyl. In still others, each of R₁ through R₄ is methyl, R₅ ismethyl, R₆ is benzyloxy methyl and R₇ is methyl, while compounds whereeach of R₁ through R₄ is methyl, R₅ is methyl, R₆ is hydroxymethyl andR₇ is methyl also find utility.

Also preferred for some embodiments, are compounds wherein each of R₁through R₄ is methyl and R₅, R₆, and R₇ form a furanyl group or where R₅is H and R₆ and R₇ form a tetrahydrofuranyl group. A further embodimentprovides compounds where R₁ through R₄ are all methyl, R₅ is H, and R₆and R₇ form a cyclopropyl ring.

It is preferred that the compositions of the invention be formulatedinto an aqueous medium, which may be delivered in topical liquid form tothe eye, via eye drops for example. Accordingly, pH and othercharacteristics of compositions of the invention are ophthalmologicallyacceptable for topical application to the eye of a patient. For someembodiments, the compound is in the form of a salt, preferably ahydrochloride or similar salt.

Since the compounds of the invention contain oxidizable hydroxylaminemoieties, which are most effective in the chemically reduced state, thecompositions preferably further comprise an anti-oxidant agent,especially a sulfhydryl compound. Exemplary compounds includemercaptopropionyl glycine, N-acetylcysteine, β-mercaptoethylamine,glutathione and similar species, although other anti-oxidant agentssuitable for ocular administration, e.g. ascorbic acid and its salts orsulfite or sodium metabisulfite may also be employed. The amount ofhydroxylamines may range from about 0.1% weight by volume to about10.0%; weight by volume and preferred is about 0.25%-weight by volume toabout 5.0% weight by volume.

The invention can also be seen to provide ophthalmologic compositionscomprising an ophthalmologically acceptable carrier or diluent togetherwith a compound having an N-hydroxypiperidine portion bound to asolubility modifying portion. In this way, the active moiety,hydroxylamine, can be delivered to the lens of an eye in need oftreatment in a “stealth” form, that is, in the form of a chemicalcompound that can have the hydroxylamine portion cleaved from thebalance of the molecule. The compound is broken down in the eye to giverise to the active hydroxylamine species for effective treatment ofcataracts or macular degeneration. The compound thus provided has asolubility in water at 25° C. of at least about 0.1% by weight and awater—n-octanol partition coefficient at 25° C. of at least about 3. Inaccordance with preferred embodiments, the water solubility is greaterthan about 0.5% by weight, preferably greater than about 2.0% and thepartition coefficient is greater than about 5, preferably greater thanabout 10.

Accordingly, it is desired that the compounds used be such that, uponadministration topically to the eye, they penetrate the cornea and areconverted to the desired hydroxylamine, preferably, anN-hydroxypiperidine. It is preferred that this conversion occurs throughenzymatic cleavage of the compound. In one preferred embodiment, thehydroxylamine portioncomprises—1,4-dihydroxy-2,2,6,6-tetramethylpiperidine.

The invention also provides methods for identifying pharmaceuticals thatcan be delivered to the lens of a patient in the form of eye drops.These methods comprise selecting a compound having a water solubility at25° C. of at least about 0.1% by weight and a water/n-octanol partitioncoefficient of at least about 5 at 25° C., which compound isenzymatically cleavable under conditions obtaining in the eye of apatient to give rise to a proximate drug for treatment of a condition ofthe eye, preferably the lens. Preferably, the active pharmaceuticalspecies is a hydroxylamine, especially one having an N-hydroxypiperidinenucleus.

The pharmaceutical compositions of the present invention may also beused for treatment of parts of the eye other than the lens. Thus, theyare suitable for ameliorating or arresting the development of maculardegeneration.

The invention includes methods for ameliorating—either slowing orarresting entirely—the development of a cataract in the lens of apatient. Some of these methods may be used to treat macular degenerationin the eye of a patient. Such methods comprise administering to the eyean ophthalmologic composition comprising an ophthalmologicallyacceptable carrier or diluent in the form of eye drops containing acompound having one or more of the foregoing compounds as an activeingredient therein. It is preferred that the administration takes placea plurality of time and, in certain preferred embodiments, chronic,periodic administration is performed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts aqueous humor levels of Tempol-H(1,4-dihydroxy-2,2,6,6-tetramethylpiperidine) in rabbit eyes treatedtopically with Compound 1 of the invention or with Tempol-H.

The present invention provides compounds and compositions that can beadministered topically to the eyes of patients who are developing or whoare at risk of developing cataracts or macular degeneration. While suchcompounds may be seen to include as a chemical fragment, hydroxylaminespecies previously known to be effective in retarding cataractdevelopment, the achievement of compounds that can be topically appliedis a very significant advance in the therapeutic arts. Indeed, theNational Institutes of Health, assignee of the Zigler patent; tried, butfailed to identify compounds that could be efficacious in therapies forcataracts or macular degeneration through topical application. In thiscontext, it is noted that the Zigler patent recites administration ofcertain compositions such as TEMPOL-H via injection and recognizes thedesirability of topical administration via eye drops, however, thisproposed route of administration was not found to be available inpractice. Accordingly, the present invention should be viewed as“pioneering” and as having satisfied a long—felt, but unserved need inthe art.

The present invention also provides compounds and compositions that canbe administered topically to the eyes or lens of a patient who hasdeveloped macular degeneration or who is at risk of developing maculardegeneration. There is no standard treatment for the “dry” form ofmacular degeneration although low vision rehabilitation may be availableto some extreme cases. The “wet” form maybe treated by laser surgerycoupled with low vision rehabilitation. Use of the present invention intreating macular degeneration has not been found to be available incurrent practice.

While not desiring to be bound by theory, it is believed that thecompounds of the present invention are absorbed across the cornea intothe eye where enzymatic processes cleave the N-hydroxypiperidine portionof the compound from the acid to which it was esterified. TheN-hydroxypiperidine moiety, once liberated, then performs the samefunctions with the same efficacy as demonstrated by Zigler.

The esters of the invention have not been known heretofore foradministration to the eye. They have certainly not been known for use inthe treatment of cataract. U.S. Pat. No. 5,981,548, in the name ofPaolini, et al., the content of which is incorporated herein byreference, depicts certain N-hydroxylpiperidine esters and their use asantioxidants in a number of contexts. However, Paolini does not discloseophthalmologic formulations or topical treatment of the eyes ofpatients. Paolini does disclose, however, useful syntheses for certainmolecules of this type.

Gupta et al. in U.S. Pat. No. 4,404,302, the content of which, disclosethe use of certain N-hydroxylamines as light stabilizers in plasticsformulations. Mitchell et al. in U.S. Pat. No. 5,462,946, the content ofwhich is incorporated herein by reference, discloses certain nitroxidesderiving from substituted oxazolidines for protection of organisms fromoxidative stress. U.S. Pat. No. 3,936,456, the content of which isincorporated herein by reference, in the name of Ramey et al., providessubstituted piperazine dione oxyls and hydroxides for the stabilizationof polymers. U.S. Pat. No. 4,691,015, to Behrens et al., the content ofwhich is incorporated herein by reference, describes hydroxylaminesderived from hindered amines and the use of certain of them for thestabilization of polyolefins.

The tissues, including the lens, of the anterior chamber of the eye arebathed by the aqueous humor. This fluid is in a highly reducing redoxstate because it contains antioxidant compounds and enzymes. The lens isalso a highly reducing environment, which maintains the hydroxlaminecompounds in the preferred reduced form. It may be necessary to includea reducing agent in the eye drop formulation but one skilled in the artmay not find it necessary in the present invention to dose separatelywith the reducing agent or to introduce it into the eye.

Preferred reducing agents may be N-acetylcysteine, ascorbic acid or asalt form, and sodium sulfite or metabisulfite. A combination ofN-acetylcysteine and sodium ascorbate may be used. A metal chelatorantioxidant, such as EDTA (ethylenediaminetetraacetic acid) or possiblyDTPA (diethylenetriaminepentaacetic acid) may also be added to keep thehydroxylamine in the reduced form in the eye drop formulation.

In accordance with one embodiment of the invention, the composition maybe delivered to the lens of an eye in need of treatment via polymericinserts, such as OCUSERT® or a contact lens or other object temporarilyresident upon the surface of the eye. Thus, the composition may beincorporated into a contact lens or some other similar means. Thecomposition may also be placed upon the eye in the ordinary fashion,e.g. in eye drops or washes. Alternatively, the compositions may beapplied in other ophthalmologic dosage forms known to those skilled inthe art, such as preformed or in situ formed gels or liposomes.Application of the anti-cataract compounds to the eye in these formsalso results in enzymatic degradation of the esters into the proximatehydroxylamine therapeutic.

The present invention provides compositions comprising apharmaceutically carrier or diluent and a compound having the formula:

-   where R₁ and R₂ are, independently, H or C₁ to C₃ alkyl;-   R₃ and R₄ are, independently C₁ to C₃ alkyl; and-   where R₁ and R₂, taken together, or R₃ and R₄, taken together, or    both may be cycloalkyl;-   R₅ is H, OH, or C₁ to C₆ alkyl;-   R₆ is C₁ to C₆ alkyl, alkenyl, alkynyl, or substituted alkyl or    alkenyl;-   R₇ is C₁ to C₆ alkyl, alkenyl, alkynyl, substituted alkyl, alkenyl,    cycloalkyl, or heterocycle-   or where R₆ and R₇, or R₅, R₆ and R₇, taken together, form a    carbocycle or heterocycle having from 3 to 7 atoms in the ring.    These compounds may also be used with ophthalmically acceptable    carriers for use in ophthalmic compositions.

The compounds of the present invention may also comprise anophthalmically acceptable carrier or diluent and a compound having anN-hydroxy piperidine portion bound to a solubility modifying portion,the compound having a solubility in water at 25° C. of at least about0.25% by weight and a water—n-octonal partition coefficient at 25° C. ofat least about 5. The composition may have the N-hydroxy piperidineportion cleavable from the compound under conditions found in the eye.It is foreseeable that this portion is cleaved under conditions in thelens of the eye. The N-hydroxy piperidine portion may be cleavedenzymatically. The compositions may also exist wherein the N-hydroxypiperidine portion is 1-oxyl-4-hydroxy-2,2,6,6-tetramethylpiperidyl.

The term C₁ to C_(n) alkyl, alkenyl, or alkynyl, in the sense of thisinvention, means a hydrocarbyl group having from 1 to n carbon atoms init. The term thus comprehends methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, and the various isomericforms of pentyl, hexyl, and the like. Likewise, the term includesethenyl, ethynyl, propenyl, propynyl, and similar branched andunbranched unsaturated hydrocarbon groups of up to n carbon atoms. Asthe context may admit, such groups may be functionalized such as withone or more hydroxy, alkoxy, alkylthio, alkylamino, dialkylamino,aryloxy, arylamino, benzyloxy, benzylamino, heterocycle, or YCO-Z, whereY is O, N, or S and Z is alkyl, cycloalkyl, heterocycle, or arylsubstituent.

The term carbocycle defines cyclic structures or rings, wherein allatoms forming the ring are carbon. Exemplary of these are cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. Cyclopropyl isone preferred species. Heterocycle defines a cyclic structure where atleast one atom of the ring is not carbon. Examples of this broad classinclude furan, dihydrofuran, tetrahydrofuran, pyran, oxazole, oxazoline,oxazolidine, imidazole and others, especially those with an oxygen atomin the ring. Five, six and seven membered rings with at least one oxygenor nitrogen atom in the ring are preferred heterocycles. Furanyl andtetrahydrofuranyl species are among those preferred.

It is preferred for certain embodiments that each of R₁ through R₄ belower alkyl that is C₁ to C₃ alkyl. Preferably, all these groups aremethyl for convenience in synthesis and due to the known efficacy ofmoieties having such substitution at these positions. However, othersubstituents may be used as well.

In certain embodiments, compounds are employed where R₆ is C₁ to C₆alkyl substituted with at least one C₁ to C₆ alkoxy or benzyloxy group.Preferred among these are compounds having ethoxy or benzyloxysubstituents. Among preferred compounds are those where each of R₁through R₄ is methyl, R₅ is H or methyl, R₆ is methyl substituted withbenzyloxy or C₁ to C₆ alkoxy, and R₇ is methyl or where R₆ and R₇ form acyclopropyl group as well as the compound in which each of R₁ through R₄is methyl, R₅ is methyl, R₆ is ethoxy or benzyloxy methyl, and R₇ ismethyl. An additional preferred compound is one in which each of R₁through R₄ is methyl, R₅ is methyl, R₆ is hydroxymethyl, and R₇ ismethyl.

Other useful compounds are those wherein each of R₁ through R₄ ismethyl, and R₅, R₆, and R₇ form a furanyl group, or in which R₆ and R₇form a tetrahydrofuranyl group. The compound where R₁ through R₄ ismethyl, R₅ is H and, R₆ and R₇ form a cyclopropyl ring is a furtherpreferred species are as those set forth in the examples below.

The compounds of the invention are formulated into compositions forapplication to the eye of patients in need of therapy. Thus, suchcompositions are adapted for pharmaceutical use as an eye drop or incontact lenses, inserts or the like. Accordingly, formulation ofcompound into sterile water containing any desired diluents, salts, pHmodifying materials and the like as are known to persons skilled in thepharmaceutical formulations art may be performed in order to achieve asolution compatible with administration to the eye. It may be that eyedrops, inserts, contact lenses, gels and other topical liquid forms mayrequire somewhat different formulations. All such formulationsconsistent with direct administration to the eye are comprehendedhereby.

The compositions of the invention may also have antioxidants in rangesthat vary depending on the kind of antioxidant used. The usage alsodepends on the amount of antioxidant needed to allow at least 2 yearsshelf-life for the pharmaceutical composition. One or more antioxidantsmay be included in the formulation. Certain commonly used antioxidantshave maximum levels allowed by regulatory authorities.

Reasonable ranges are about 0.01% to about 0.15% weight by volume ofEDTA, about 0.01% to about 2.0% weight volume of sodium sulfite, andabout 0.01% to about 2.0% weight by volume of sodium metabisulfite. Oneskilled in the art may use a concentration of about 0.1% weight byvolume for each of the above. N-Acetylcysteine may be present in a rangeof about 0.1% to about 5.0% weight by volume, with about 1% to about 10%of hydroxylamine concentration being preferred. Ascorbic acid or saltmay also be present in a range of about 0.1% to about 5.0% weight byvolume with about 1% to about 10% weight by volume of hydroxylamineconcentration preferred. Other sulfhydryls, if included, may be the samerange as for N-acetylcysteine. Other exemplary compounds includemercaptopropionyl glycine, N-acetyl cysteine, β-mercaptoethylamine,glutathione and similar species, although other anti-oxidant agentssuitable for ocular administration, e.g. ascorbic acid and its salts orsulfite or sodium metabisulfite may also be employed.

A buffering agent may be used to maintain the pH of eye dropformulations in the range of about 4.0 to about 8.0; this is necessaryto prevent corneal irritation. Because the compounds of this inventionare esters, the pH will need to be about 3.5 to about 6.0, preferablyabout 4.0 to about 5.5, in order to prevent hydrolysis of the ester bondand to ensure at least a 2-year shelf life, for the product. This pHalso ensures that most of the hydroxylamine is in its protonated formfor highest aqueous solubility. The buffer may be any weak acid and itsconjugate base with a pKa of about 4.0 to about 5.5; e.g. aceticacid/sodium acetate; citric acid/sodium citrate. The pKa of thehydroxylamines is about 6.0.

The compounds of the present invention may also include tonicity agentssuitable for administration to the eye. Among those suitable is sodiumchloride to make formulations of the present invention approximatelyisotonic with 0.9% saline solution.

In certain embodiments, the compounds of the invention are formulatedwith viscosity enhancing agents. Exemplary agents arehydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, andpolyvinylpyrrolidone. The viscosity agents may exists in the compoundsup to about 1.6% weight by volume. It may be preferred that the agentsare present in a range from about 0.2% to about 0.25 weight by volume. Apreferred range for polyvinylpyrrolidone may be from about 0.1% to about0.2% weight by volume. One skilled in the art may prefer any rangeestablished as acceptable by the Food and Drug Administration.

The compounds of the invention may have cosolvents added if needed.Suitable cosolvents may include glycerin, polyethylene glycol (PEG),polysorbate, propylene glycol, and polyvinyl alcohol. The presence ofthe cosolvents may exist in a range of about 0.2% to about 1.0% weightby volume. It may also be preferred that polyvinyl alcohol may beformulated in the compounds of the invention in a range of about 0.1% toabout 4.0% weight by volume. One skilled in the art may prefer rangesestablished as acceptable by the Food and Drug Administration.

Preservatives may be used in the invention within particular ranges.Among those preferred are up to 0.013% weight by volume of benzalkoniumchloride, up to 0.013% weight by volume of benzethonium chloride, up to0.5% weight by volume of chlorobutanol, up to 0.004% weight by volume orphenylmercuric acetate or nitrate, up to 0.01% weight by volume ofthimerosal, and from about 0.01% to about 0.2% weight by volume ofmethyl or propylparabens.

For effective treatment of cataract, one skilled in the art mayrecommend a dosage schedule and dosage amount adequate for the subjectbeing treated. It may be preferred that dosing occur one to four timesdaily for as long as needed. The dosage amount may be one or two dropsper dose. The dosage schedule may also vary depending on the active drugconcentration, which may depend on the hydroxylamine used and on theneeds of the patient. It may be preferred that the active amount be fromabout 0.1% to about 10.0% weight by volume. In some embodiments, it ispreferable that the active drug concentration be 0.25% to about 5.0%weight by volume.

An ophthalmologist or one similarly skilled in the art may have avariety of means to monitor the effectiveness of the dosage scheme andadjust dosages accordingly. Effectiveness may be determined by theophthalmologist by observing the degree of opacity of the lens atintervals by slit-lamp examination, or other means and increasing thefrequency and/or concentration of the eye drop prescribed, if needed.

Some embodiments of the invention are methods of administering anantioxidant to a mammal comprising contacting the mammal with acomposition comprising a pharmaceutically acceptable carrier or diluentand a compound having an N-hydroxy piperidine portion bound to asolubility modifying portion, the compound having a solubility in waterat 25° C. of at least about 0.25% by weight and a water—n-octonalpartition coefficient at 25° C. of at least about 5. In otherembodiments, the methods may identify a pharmaceutical for delivery tothe eye of a patient in the form of eye drops comprising selecting acompound having a water solubility at 25° C. of at least about 0.25% byweight and a water—n-octonal partition coefficient of at least about 5at 25° C., which compound is enzymatically cleavable under conditionsobtained in the lens of the eye of a patient to give rise to anN-hydroxy piperidine.

The present invention has optimal use in ameliorating the development ofa cataract in the eye of a patient. Another optimal use includes thetreatment of macular degeneration in the retina of a patient. Theophthalmic compositions of the present invention may be utilized byadministration to the eye of a patient affected by these maladies. Thisadministration may be performed by eye drops, in eye washes, or viaother acceptable delivery means known to those skilled in the art suchas, dispersion or delivery by contact lens. Other forms ofadministration of the compositions of the present invention, wherein thedelivery to the eye is not called for, may include oral tablets, liquidsand sprays; intravenous, subcutaneous and intraperitoneal injections;application to the skin as a patch or ointment; enemas, suppositories,or aerosols.

A variety of esterases is known to be present in ocular tissues,especially the cornea. The specific esterase(s) that cleaves the estersof the present series is not identified. The cleavage of the estersoccurs rapidly and essentially completely on administering the compoundsto the eyes of rabbits. This is shown by the presence of tempol-H in theaqueous humor at all times (30, 60, 90 and 120 minutes) examined aftertopical dosing. In contrast, the esters are stable in aqueous solutions;e.g. solution of Ester 4 at 40° C., in acetate buffer at pH 4.6, isstable for 3 months.

It may be preferred that at least 0.1% solubility is needed for an eyedrop, even for a suspension formulation. Completely water-insolublecompounds may not be effective. Esters that are soluble in water (>0.1%weight by volume) are preferred. Esters with less than 0.1% solubilitymay be used in the form of suspensions or ointments or otherformulations. Solubility is determined by mixing 100 mg of test compoundwith 1 ml of water, at room temperature and adding additional 1 mlquantities of water, with mixing, until ester dissolves completely.

Corneal penetration is shown by measuring a substantial concentration(e.g. >5 μM) of the effective hydroxylamine and/or ester in the aqueoushumor after administering a solution of the compound in vivo to the eyesof rabbits. This is determined by electron spin resonance (ESR), highperformance liquid chromatography (HPLC) or gas chromatography (GC)assay of the rabbit aqueous humor. In vitro corneal penetration methodsmay also be used prior to the in vivo testing method particularly forscreening compounds.

Esters are selected for these tests based on their calculated ormeasured octanol/water partition coefficient (P). Hydrophilic compoundssuch as tempol-H cannot penetrate the lipophilic epithelial layer of thecornea. Partition coefficients of tempol-H and esters that penetrate areas follows:

P (Calculated)* Tempol-H  0.8 (measured, 0.5) Ester 4 16.4 Ester 8  8.2Ester 14  6.3 *Clog P version 4.0, Biobyte Corporation

Enzymatic conversion is essentially complete at greater than 90%hydrolysis of the ester in vivo to the alcohol and acid afteradministering the compound to the eye of rabbits. The conversion may bedetermined by HPLC or GC assay of the rabbit aqueous humor.

Alternatively, the enzymatic conversion may be determined by incubatingthe compound in plasma or corneal homogenate and assaying samplesperiodically by HPLC or GC to monitor the rate of breakdown. Esters witha half-life of less than about 1 or 2 hours are candidates. This methodmay be the preferred screening procedure before in vivo testing.

Esters should have less than about 10% hydrolysis at 40° C., after 3months, in aqueous solution at pH 4.0-5.0. This extrapolates to a shelflife of the ester in solution of at least 18 months at room temperature,which may be preferred for an eye drop product.

The compounds of this invention may have uses in fields broader thanophthalmology. These areas may include, for example, protection of hairfollicles and rectum from radiation damage during radiation therapy forcancer and amelioration of irritation and inflammation during lasersurgery of the eye, including trabeculectomy treatment for glaucoma andkeratectomy for corneal reshaping.

While the present invention has been particularly shown and describedwith reference to the presently preferred embodiments thereof, it isunderstood that the invention is not limited to the embodimentsspecifically disclosed herein. Numerous changes and modifications may bemade to the preferred embodiment of the invention, and such changes andmodifications may be made without departing from the spirit of theinvention. It is therefore intended that the appended claims cover allsuch equivalent variations as they fall within the true spirit and scopeof the invention.

EXAMPLES

The present invention is illustrated in certain embodiments by referenceto the following examples. The examples are for purposes of illustrationonly and are not intended to be limiting in any way.

Example 1

Determination of Ester Compound Stability In Aqueous Solution. Method: A0.1-0.5% solution of the ester compound was prepared in buffer (pH4.5-5.0) containing DTPA or EDTA. The solution was filled into amberglass vials, which were sealed and placed in a controlled temperaturecontainer maintained at 40° C. Sample vials were removed periodicallyand stored at 0-5° C. until analyzed by HPLC, GC, or GC/MS analyticalmethods, and found to be stable after 3 months under these conditions.

To be useful as an anti-cataract drug the agent must penetrate into thelens. This may be included in the method for selecting an anti-cataractcompound. A description of method for tempol-H follows:

Example 2 Drug Penetration of Organ Cultured Rat Lenses

In contrast to drugs tested previously as anti-cataract agents, tempol-Hand tempol have a remarkable ability to penetrate lens tissue from thesurrounding fluid. The experiments described in this section determinedthe time course, active compound concentrations and compounddistribution in the lens, after incubation with rat lenses under theorgan culture conditions.

Method: Rat lenses were cultured as follows: Rat lenses were obtainedfrom Sprague-Dawley rats. The lenses were incubated in 24-well clusterdishes in modified TC-199 medium and were placed in a 37° C. incubatorwith a 95% air/5% CO₂ atmosphere. The lenses were incubated in 2 ml ofculture medium, which was adjusted to 300 milliosmoles (mOsm). Lenseswere incubated, for 1 to 24 hours, in the culture medium with 4.0 mMtempol-H, or with 4.0 mM of the oxidized form, tempol. At theappropriate time, the lenses were removed from the medium, blotted dry,homogenized and were analyzed for active compound by electron spinresonance method (ESR). In one experiment, lenses were incubated for 4hours and dissected into epithelial, cortical and nuclear sectionsbefore analysis.

Results: Concentrations (mM, in lens water) of tempol-H reached 0.4 mM,0.8 mM and 1.0 mM, respectively, after 1, 2 and 4 hours incubation ofactive compound. Levels of tempol-H found, after incubation of lenseswith the oxidized form tempol, reach 0.6 mM, 1.5 mM and 2.8 mMrespectively. In the latter case, only a trace (5% or less) of theoxidized form tempol, was found in the lens; it was almost completelyconverted to the reduced form tempol-H.

Distribution of tempol-H between the lens epithelium, cortex and nucleuswas fairly even, after a 4-hour incubation period with tempol-H. Levelsof tempol-H reached 1.5 mM, 0.8 mM and 1.0 mM, respectively, in theepithelium, cortex and nucleus. Levels of tempol-H/tempol in lensesincubated with the oxidized form, tempol, were 1.2 mM, 2.9 mM and 2.0mM, respectively. In the latter case, all compounds in the nucleus werein the reduced form with only about 5% in the epithelium in the oxidizedform.

Conclusion: Both the reduced and oxidized forms of the active agentreadily penetrated into the cultured rat lens from the bath medium anddistributed to the epithelium, cortex and nucleus. Incubation of lenseswith the oxidized form tempol, results in high concentrations of reducedcompound tempol-H throughout the lens.

Example 31-oxyl-4-(3′-ethoxy-2′,2′-dimethyl)propanecarbonyloxy-2,2,6,6-tetramethylpiperidine

1,1′-carbonyldiimidazole was added in small portion (1.27 g, 7.84 mmol)to a stirred solution of 3-ethoxy-2,2-dimethylpropionic acid (750 mg,7.13 mmol; prepared according to the procedure described in J. Org.Chem., 38, 2349, 1975, the content of which was incorporated herein byreference) in dry DMF (10 mL). A vigorous gas evolution was observed.This solution was heated at 100° C. for 1 h. To this mixture was thenadded tempol (900 mg, 5.23 mmol) and 1,8-diazabicyclo [5,4,0]undec-7-ene(DBU) (800 mg, 5.26 mmol) and continue heating for 12 h. The reactionmixture was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate (100 mL) and was washed successively with 1NHCl, saturated NaHCO₃ and brine, was dried over anhydrous sodium sulfateand was concentrated in vacuo to give red colored solid (1.48 g). Thiswas purified by column chromatography on silica gel usingcyclohexane:ethyl acetate (8:1) as eluent to give a red coloredcrystalline solid (1.22 g, 70.0%).

IR (KBr, cm−1): 1360 (N—O.), 1725 (ester)

Example 41-hydroxy-4-(3′-ethoxy-2′,2′-dimethyl)propanecarbonyloxy-2,2,6,6-tetramethylpiperidineHydrochloride

The nitroxide of Example 2 (1.02 mg, 3.34 mmol) was added to a solutionof saturated hydrogen chloride in ethanol (20 mL). The red colordisappears quickly and the resulting yellow colored solution was boiledto give a clear colorless solution. The solution was concentrated invacuo, was dissolved in 100 mL ethyl acetate and was washed withsaturated NaHCO₃ to obtain the hydroxylamine free-base. The ethylacetate layer was separated and concentrated to give a red colored oilwhich was mostly nitroxide, by TLC. This oil was purified by columnchromatography on silica gel using cyclohexane:ethyl acetate (4:1) aseluent to give a red colored crystalline solid (700 mg). The solid wasdissolved in a solution of saturated hydrogen chloride in ethanol (20mL), was concentrated in vacuo, and was recrystallized from ethylacetate:diisopropylether (2:1, 50 mL) to give white crystalline solid(320 mg).

m.p. 140-142° C. (dec.). ¹H-NMR (270 MHz, D₂O) ppm: 1.48 (6H, s); 1.57(3H, t); 1.63 (12H, s); 1.82 (2H, s); 2.02 (2H, t); 2.40 (2H, d), 3.88(2H, q); 5.44 (1H, m) IR (KBr, cm−1): 3487 (OH), 1726 (ester) Mass Spec.(EI, m/z) 301 (M+)

Example 5a1-oxyl-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine

A suspension of sodium hydride (60% in oil, 1.0 g, 25 mmol) in dry THF(50 mL) was stirred at room temperature for 5 min and to this mixturewas added tempol (4.0 g, 23 mmol). The mixture was stirred for 1 h,cyclopropanecarbonyl chloride (2.4 g, 23 mmol) was added dropwise over 5min and then it was refluxed for 1 h. The reaction mixture wasconcentrated under reduced pressure. The residue was taken up in pentane(100 mL) and the supernatant was separated and concentrated underreduced pressure to give red solid. This solid was purified by columnchromatography on silica gel using cyclohexane:ethyl acetate (3:1) aseluent to give a red colored crystalline solid (1.4 g, 5.8 mmol, 25.3%).

IR (KBr, cm−1): 1361 (N—O.), 1720 (ester)

Example 5b AlternativeMethod—1-oxyl-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine

1,1′-Carbonyldiimidazole (1.78 g, 11 mmol) was added in small portionsto a stirred solution of cyclopropanecarboxylic acid (860 mg, 10 mmol)in dry DMF (10 mL). A vigorous gas evolution was observed. This solutionwas heated at 40° C. for 1 h. To this mixture was then added tempol(1.72 g, 10 mmol) and 1,8-diazabicyclo[5,4,0]undec-7-ene(DBU) (1.52 g,10 mmol) and it was heated at 40° C. for another 12 h. The reactionmixture was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate (100 mL) and was washed successively with 1NHCl, saturated NaHCO₃ and brine. The ethyl acetate layer was separated,dried over anhydrous sodium sulfate and concentrated in vacuo to givered colored solid. This solid was purified by column chromatography onsilica gel using cyclohexane:ethyl acetate (8:1) as eluent to give a redcolored crystalline solid (720 mg, 30.0%).

IR (KBr, c m⁻¹): 1360 (N—O.), 1720 (ester)

Example 5c AlternativeMethod—1-oxyl-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine[DCC/DMAP Esterification Method]

To a stirred solution of Tempol (1.72 g, 0.01 mmole),cyclopropanecarboxylic acid (0.946 g, 0.011 mmole), and DMAP (0.12,0.001 mmole) in dichloromethane (25 ml) was added DCC (2.27 g, 0.11mmole) and the mixture was stirred overnight at room temperature. Themixture was filtered over celite and the solution was evaporated underreduced pressure. The product was isolated by silica gel columnchromatography using first hexane and then 10% ethyl acetate in hexane.Yield: 2.26 g (94.1). IR and NMR were consistent with the assignedstructure.

Example 61-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidineHydrochloride (Compound 1)

The nitroxide of Example 5a (2.2 g, 9.15 mmol) was added to a solutionof saturated hydrogen chloride in ethanol (20 mL). The red colordisappeared quickly and the resulting yellow colored solution was boiledto give clear colorless solution. The solution was concentrated invacuo, dissolved in 100 mL ethyl acetate and was washed with saturatedNaHCO₃ to obtain the hydroxylamine free-base. The ethyl acetate layerwas separated, acidified with ethereal HCl, and concentrated to givewhite solid, which was recrystallized from ethanol (10 mL) as a whitecrystalline solid 1.15 g (4.13 mmol, 45.1%). m.p. 224-228° C. (dec.).

¹H-NMR (270 MHz, D₂O) ppm: 0.97 (4H, d); 1.43 (1H, m); 1.44 (6H, s),1.46 (6H, s); 1.90 (2H, t); 2.28 (2H, t); 5.2(1H, m) IR (KBr, cm−1):3478 (OH), 1720 (ester) Mass Spec. (EI, m/z) 240 (M+)

Example 71-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidineHydrochloride (Alternate Method)

The nitroxide of Example 5a (700 mg, 2.91 mmol) was added to a solutionof saturated hydrogen chloride in ethanol (20 mL). The red colordisappeared quickly and the resulting yellow colored solution was boiledto give a clear colorless solution. The solution was concentrated invacuo, dissolved in 100 mL ethyl acetate and concentrated to half volumeto give a white crystalline solid, 627 mg (2.25 mmol, 77.5%.). m.p.224-227° C. (dec.).

¹H-NMR (270 MHz, D₂O) ppm: 0.97 (4H, d); 1.43 (1H, m); 1.44 (6H, s),1.46 (6H, s); 1.90 (2H, t); 2.28 (2H, t); 5.2(1H, m) IR (KBr, cm−1):3476 (OH), 1720 (ester) Mass Spec. (EI, m/z) 240 (M+)

Example 81-oxyl-4-(3′-benzyloxy-2′,2′-dimethyl)propanecarbonyloxy-2,2,6,6-tetramethylpiperidine

To a stirred solution of 3-benzyloxy-2,2-dimethylpropionic acid (1.04 g,5 mmol), (prepared by a method similar to that described in J. Org.Chem., 38, 2349, 1975), in dry DMF (5 mL), was added1,1′-carbonyldiimidazole in small portions. A vigorous gas evolution wasobserved. This solution was heated at 50° C. for 30 min. To this mixturewas then added tempol (900 mg, 5.23 mmol) and1,8-diazabicyclo[5,4,0]undec-7-ene(DBU) (800 mg, 5.26 mmol). The mixturewas heated at 50° C. for 3 days (monitored by TLC) and then it wasconcentrated under reduced pressure. The residue was dissolved in ethylacetate (100 mL), washed successively with 1N HCl, saturated NaHCO₃ andbrine, and dried over anhydrous sodium sulfate. The dried solution wasconcentrated in vacuo to give red colored solid (1.48 g). This solid waspurified by column chromatography on silica gel using cyclohexane:ethylacetate (3:1) as eluent to give a red colored crystalline solid (1.02 g,2.8 mmol, 56.2%).

IR (KBr, cm−1): 1359 (N—O.), 1732 (ester)

Example 91-hydroxy-4-(3′-benzyloxy-2′,2′-dimethyl)propanecarbonyloxy-2,2,6,6-tetramethylpiperidineHydrochloride

The nitroxide of Example 8 (1.02 mg, 3.34 mmol) was added to a solutionof saturated hydrogen chloride in ethanol (20 mL). The red colordisappears quickly and the resulting yellow colored solution was boiledto give clear colorless solution. The solution was concentrated in vacuoand the residue dissolved in ethyl acetate (20 mL). Hexane (20 mL) wasadded and product began to oil out; the mixture was then allowed tostand for 12 h. An oily residue was obtained by decantation of thesolvent and it was treated with was isopropyl ether and warmed. Uponcooling the mixture, a waxy solid was obtained and recrystallized fromethyl acetate to give white crystalline solid (0.6 g, 1.5 mmol, 45%).

¹H-NMR (270 MHz, D₂O) ppm: 1.26 (6H, s), 1.51 (6H, s); 1.65 (6H, s);2.01 (2H, t); 2.44 (2H, d), 5.40 (1H, m); 3.46 (2H, s), 4.55 (2H, S),7.31 (5H, s) IR (KBr, cm−1): 3480 (OH), 1712 (ester), 710 (aromatic)Mass Spec. (EI, m/z) 262 (M+)

Example 101-hydroxy-4-(3′-hydroxy-2′,2′-dimethyl)propanecarbonyloxy-2,2,6,6-tetramethylpiperidineHydrochloride

Pd/C (5%, 100 mg) was added to a solution of1-oxyl-4-(3′-benzyloxy-2′,2′-dimethyl)propanecarbonyloxy-2,2,6,6-tetramethylpiperidine(1.0 g, 3.83 mmol) in ethanol, and the mixture was hydrogenated in aPaar hydrogenation apparatus at 45 psi for 12 h. The reaction mixturewas filtered through celite and concentrated in vacuo to give a clearcolorless oil, which was purified by column chromatography on silica gelusing cyclohexane:ethyl acetate (3:1) as eluent to give a colorless oil.The oil was dissolved in a solution of saturated hydrogen chloride inethanol (20 mL) and concentrated in vacuo. Product crystallized uponstanding, and was recrystallized from ethanol (123 mg, 0.4 mmol, 10.4%).m.p. 210-215° C. (dec.).

¹H-NMR of the free base (270 MHz, CDCl₃) ppm: 1.14 (6H, s), 1.44 (6H,s); 1.57 (6H, s); 1.70 (2H, m); 2.8 (1H, s, br), 3.65 (2H, s) 5.16 (1H,m) IR (KBr, cm−1): 3480 (OH), 1712 (ester), 710 (aromatic) Mass Spec.(EI, m/z) 262 (M+)

Example 111-oxyl-4-(1-methyl-cyclopropane)carbonyloxy-2,2,6,6-tetramethylpiperidine

A suspension of sodium hydride (60% in oil 2.2 g), in dry THF (80 mL)was stirred at room temperature for 5 min and then tempol (3.0 g, 17.44mmol) was added. The mixture was stirred for 30 min,1-methyl-cyclopropanecarbonyl chloride (2.2 g, 18.71 mmol) was addeddrop wise over 5 min and then it was refluxed for 12 h. The reactionmixture was concentrated under reduced pressure and the residuecrystallized immediately. The product was purified by columnchromatography on silica gel using cyclohexane:ethyl acetate (3:1) aseluent to give a red colored crystalline solid (2.0 g, 7.86 mmol,45.1%).

IR (KBr, cm−1): 1314 (N—O.), 1722 (ester)

Example 121-hydroxy-4-(1-methyl-cyclopropane)carbonyloxy-2,2,6,6-tetramethylpiperidineHydrochloride

The nitroxide of Example 11 (700 mg, 2.91 mmol) was added to a solutionof saturated hydrogen chloride in ethanol (10 mL). The red colordisappears quickly and the resulting yellow colored solution was boiledto give a clear colorless solution. The solution was concentrated invacuo to give white crystalline solid, which was filtered, washed withethyl acetate and dried in vacuo (0.700 mg, 2.4 mmol, 82.7%)

m.p. 215° C.-220° C. (dec.). ¹H-NMR (270 MHz, D₂O) ppm: 0.80 (2H, d);1.19 (2H, m); 1.21 (2H, s); 1.44 (15H, s); 2.03 (4H, m); 5.10 (1H, m)Mass Spec. (EI, m/z) 254 (M+)

Example 13 1-oxyl-4-(2-furan)carbonyloxy-2,2,6,6-tetramethylpiperidine

A stirred mixture of sodium methoxide (25% sodium methoxide in methanol,200 mg) in benzene (100 mL) was heated to reflux and the benzene wasgradually distilled off to half volume to obtain a fine suspension ofsolid sodium methoxide. To this mixture was added tempol (1.76 g, 10mmol), methyl 2-furoate (1.26 g, 10 mmole) and benzene (50 mL).Distillation of benzene was continued for 8 h to remove formed methanol.The volume of benzene in the flask was maintained by adding morebenzene. The benzene layer was washed with 1 N HCl, then with water,dried over anhydrous sodium sulfate and evaporated to dryness to give ared solid (1.72 g), which was recrystallized from hexane to give 1.45 gof product. It was further purified by column chromatography on silicagel using cyclohexane:ethyl acetate (3:1) as eluent to give a redcolored crystalline solid (1.02 g, 3.82 mmol, 32.8%).

IR (KBr, cm−1): 1364 (N—O.), 1716 (ester), 706 (aromatic)

Example 141-hydroxy-4-(2′-furan)carbonyloxy-2,2,6,6-tetramethylpiperidinehydrochloride

The nitroxide of Example 13 (300 mg, 1.13 mmol) was added to a solutionof saturated hydrogen chloride in ethanol (10 mL). The red colordisappeared quickly and the resulting yellow colored solution was boiledto give clear colorless solution. The solution was kept at roomtemperature for 1 h and a white crystalline solid separated. It wasfiltered, washed with ethyl acetate and dried in vacuo to afford thehydroxylamine (220 mg, 0.72 mmol, 64.5%, m.p. 209.4° C.-210.4° C.).

¹H-NMR (270 MHz, D₂O) ppm: 1.49 (6H, s); 1.62 (6H, s); 2.03 (2H, t);2.42 (2H, d), 5.49 (1H, m); 6.63 (1H, q); 6.64 (1H, d), 7.34 (1H, d),7.74 (1H, s) Mass Spec. (EI, m/z) 266 (M+)

Example 151-oxyl-4-(3′-tetrahydrofuran)carbonyloxy-2,2,6,6-tetramethylpiperidine

To a stirred solution of 3-tetrahydrofuancarboxylic acid (1.5 g, 13mmol) in dry DMF (20 mL) was added 1,1′-carbonyldiimadazole (2.3 g,14.18 mmol) in small portions. A vigorous gas evolution was observed.This solution was heated at 70° C. for 1 h. To this mixture was thenadded tempol (2.23 g, 12.97 mmol) and1,8-diazabicyclo[5,4,0]undec-7-ene(DBU) (2.0 g, 13.14 mmol) and heatingwas continued for 12 h. The reaction mixture was poured into 250 mLwater and extracted with ether (2×100 mL). The ethereal layers werecombined and washed successively with 1N HCl, saturated NaHCO₃ andbrine, dried over anhydrous sodium sulfate and concentrated in vacuo togive red colored solid (2.05 g), that recrystallized from ethylacetate:Hexane (1:2) to obtain pure red crystalline solid nitroxide(1.45 g, 5.36 mmol, 37.8%).

IR (KBr, cm−1): 1360 (N—O.), 1725 (ester)

Example 161-hydroxy-4-(3′-tetrahydrofuran)carbonyloxy-2,2,6,6-tetramethylpiperidinehydrochloride

The nitroxide of Example 15 (300 mg, 1.11 mmol) was added to a solutionof saturated hydrogen chloride in ethanol (10 mL). The red colordisappeared quickly and the resulting yellow colored solution was boiledto give a clear colorless solution. The solution was kept at roomtemperature for 1 h and a white crystalline solid separated. The solidwas filtered, washed with ethanol and dried in vacuo to afford product(146 mg, 0.48 mmol, 42.86%, m.p. 221.0° C.-223.2° C.).

¹H-NMR (270 MHz, DMSO-d₆) ppm: 0.84 (2H, m); 0.90 (2H, m); 1.35 (6H, s);1.46 (6H, s); 1,65 (1H, m); 2.13 (2H, t); 2.44 (2H, d), 5.14 (1H, m)

Example 17 Absorption of Representative Compounds Across the Corneas ofAnimals

Groups of six New Zealand White rabbits were used in the study toevaluate the absorption of tempol-H and compound 1. The test compoundswere prepared in sterile saline solutions at a concentration of 3.5%weight by volume. The animals were held in restraining boxes duringinstillation of eye drops, 50 μL in each eye, using a micropipette.After dosing, the eye was gently held closed for 60 seconds. The rabbitswere dosed twice daily for 4 consecutive days. On the fifth day, rabbitswere dosed once and then euthanized at 30 minutes post dose (2 rabbits),60 minutes post-dose (2 rabbits) and at 120 minutes post-dose (2rabbits). Immediately after euthanization, aqueous humor was collectedfrom each rabbit. The aqueous concentration of tempol-H in each samplewas measured using the electron spin resonance (ESR) method.

Aqueous humor levels of tempol-H after dosing with tempol-H, were belowdetectable limits of the assay at all time points (see FIG. 1). Aqueoushumor concentrations of tempol-H after dosing with compound 1 weremaximal at 30 minutes post-dose but were still present at 2 hourspost-dose. (see FIG. 1 and Table 1).

TABLE I Aqueous Humor Concentrations: Absorption Studies in RabbitsDose: 50 μL of 3.5% solution, single dose (N = 4 eyes/timepoint)Concentration of Tempol-H μM 30 minutes 60 minutes 120 minutes Compound1 51.0 20.0 1.5 30.0 30.0 1.2 18.0 6.0 7.0 30.0 3.0 8.0 Mean 32.3 14.84.4 μg/ml (5.5) (2.5) (0.75)

Example 18 Identification of Metabolites of Compound 1 in Rabbit Eye

Aqueous humor samples, from the in vivo rabbit study described inExample 16 were identified by GC/MS for the presence of compound 1 andits metabolites, tempol-H and carboxylic acid (R₁COOH), formed byhydrolysis of compound 1 by ocular esterases. Both the metabolites wereobserved but not Compound 1. This confirmed that Compound 1 wascompletely converted to its metabolites.

A sample of aqueous humor was freeze dried in a 10 mL amber coloredglass vial containing a tiny magnetic bar. To this was added 1 mL ofmethylene chloride and the solution was stirred for two minutes andallowed to stand for five minutes. A 3 μL aliquot of the methylenechloride layer was injected into the GC column. Thecyclopropanecarboxylic acid was detected by a mass spectrometer detectorat 13.02 (retention time) with m/z=85 (GC model 5989B and MS model 5890series II (both made by HP)). Agilent DB-5 column 25 m length, 0.2 mmdiameter was used. Carrier gas He at 22 cm/sec. Inlet temperature was250° C., detector 280° C. For every injection, the temperature was heldat 35° C. for 5 minutes, then was increased to 240° C. at 10° C./min,and was held at 240° C. for 3 minutes. Splitless injection was used.

Example 19

Tolerance of compound 1 in vivo in Rabbit Eyes Eyedrops containing 3.5%compound 1 were administered six times, at 1-hour intervals, to each eyeof two conscious rabbits. The drug was well tolerated and no adversefindings were noted in this preliminary study.

Example 20 Ocular Bioavailability in Rabbit

The ocular bioavailability of compounds 2 and 3 was evaluated in NewZealand White rabbits. Each compound was dissolved in 10 mM phosphatebuffer, pH 7.0 to a concentration of 125 mM. This concentration wasequal to ˜3.5% for compounds 2 and 3. Fifty μl was instilled onto thecornea of both eyes of each rabbit 6 times at 1-hour intervals. Tworabbits were used for each compound. One rabbit treated with eachcompound was euthanized 30 minutes after the last dose and the secondwas euthanized 90 minutes after the final dose.

After death, the eyes of each rabbit were immediately enucleated and ablood sample was collected from the orbit. Aqueous humor was collectedfrom each eye with a syringe and then the lens was dissected from theeye. The capsule/epithelium was carefully separated from the fiber massand both parts were frozen on dry ice, the capsule/epithelium in 100 μlof 5 MM DTPA (diethylenetriaminepentaacetic acid) solution and the fibermass in a sealed vial without added liquid. Likewise, the aqueous andblood samples were quick frozen. The rest of each eye including thecornea, retina, sclera and vitreous were frozen for possible futuredissection and analysis. All samples were transported to the lab on dryice and were stored at −75° C. until processed.

The aqueous concentration of tempol-H in each sample was measured usingthe electron spin resonance (ESR) method. Analysis of the aqueous humorreveals that both compounds penetrated the cornea and entered theaqueous chamber. The highest concentrations for both compounds waspresent in the 30-minute sample with the 90-minute samples beingsignificantly reduced in concentration. Small amounts (2-3 μM of eachcompound) were also detected in the blood.

Example 21 Aqueous Humor Concentrations of Compounds 2 and 3; in Rabbits

TABLE II Dose of Compound 2 and 3: 50 μL of 125 mM solution, at hourlyintervals × 6 (N = 2 eyes/timepoint) Concentration of Tempol-H μM 30minutes 60 minutes Blood Compound 3 31.4 11.6 2.3 22.2 9.0 2.5 Mean 26.810.8 2.4 μg/ml (4.6) (1.9) (0.4) Compound 2 52.4 6.0 3.6 35.2 5.7 0.6Mean 43.8 5.9 2.1 μg/ml (7.5) (1.0) (0.4)

Example 22 Aqueous Solubility Data

TABLE III Solubility of Compound of Example 6 was determined at roomtemperature in various systems. Solubility Solubility Conditions mg/ml %w/v Water 74.9 7.5 0.9% Sodium chloride 40.5 4.1 0.01 M Acetate bufferat pH 4.8 68.6 6.9 0.01 M Citrate buffer at pH 4.8 71.1 7.1 Water + 1%w/v glycerin 62.2 6.2 Water + 1% w/v propylene glycol 63.8 6.4

Similarly, the solubility compounds of Examples 10 and 16 in water weredetermined to be >3.5% w/v (>35 mg/ml) in water whereas the compound ofExample 12 is soluble at approximately 0.1% w/v in water.

TABLE IV Partition Coefficient of Ester Compounds

Examples R Calculated PC 23 H 0.8 24

7.2 25

16.2 26

50.1 27

53.7 28

125.9 29

91.2 30

6.3 31

114.8 32

34.7 33

199.5 34

4.3 35

8.1 36

144.5 37

10 38

51.3 39

575.4 40

34.7 41

69.4 42

67.6 43

39.8

TABLE V Melting Points of Ester Compounds

Examples R1 R2 M.P. (° C.) 44 O

97.2-98.2 44 OH (as HCl salt)

224-228° C. (dec.). 45 OH (as HCl salt)

224-227° C. (dec.). 46 O

103.9-105.2 47 OH (as HCl salt)

209.4-210.1 48 O

150-152.3 49 OH (as HCl salt)

250.6-253.2 50 O

64.8-66.1 51 OH (as HCl salt)

229.0-230.9 52 O

107-109.3 53 OH (as HCl salt)

220.0-223.0 54 O

111.1-112.3 55 OH (as HCl salt)

228.0-231.2 56 O

121.2-122.9 57 OH (as HCl salt)

241.8-244.6 58 O

145.2-146.4 59 OH (as HCl salt)

237.8-269.1 60 O

132-133.0 61 OH (as HCl salt)

267.9-270 62 O

68.3-69.9 63 OH (as HCl salt)

264.8-266.3

Spectral Data for the Ester Compounds

¹H-NMR (270 MHz, DMSO-d₆) ppm: spectral data that was common to all4-substituted-1-hydroxy-2,2,6,6-tetramethylpiperidine hydrochlorideportion 1.35 (6H, s); 1.46 (6H, s); 2.13 (2H, t); 2.44 (2H, d), 5.14(1H, m)

IR CKBr) cm-1 ¹H-NMR (270 MHz, DMSO-d₆) ppm: for Examples Carbonyl(s)the ester moiety 57 1716 3.75 (S, 9H); 6.95 (s, 2H) 61 1738 2.79 (t,2H); 3.31 (t, 2H); 7.45 (m, 2H); 7.55 1687 (m, 1H); 7.93 (d, 2H) 53 16826.87 (d, 2H); 7.83 (d, 2H), 10.3 (br, s, 1H) 51 1718 3.9 (s, 3H), 7.18(d, 2H), 8.07 (d, 2H0 1755 59 1723 6.54, 7.85 (dd, J=16.0 Hz); 6.84 (m,1H); 7.24 (m, 1H); 7.54 (d, 1H); 7.86 (d, 1H); 10/2 (br, s, 1H) 61 17181.96 (m, 2H); 2.30 (m, 4H); 2.78 (m, 2H) 49 1688 2.88 (S, 6H); 6.65 (d,2H); 7.73 (d, 2H) 53 1682 3.70 (s, 3H); 7.20 (d, 2H); 7.72 (d, 2H)

The following Tables VI to XII describe methods used in the synthesis ofadditional examples of the ester compounds of the invention. Theappropriate carboxylic acid listed in the Tables is converted to theester nitroxide by the DCC/DMAP esterification method of Example 5c. Theester nitroxide is converted to the corresponding 1-hydroxypiperidine bythe methods described in Examples 6 and 7.

3-acyloxy-2.2-dimethylpropionic acids were prepared by the methoddescribed in U.S. Pat. No. 4,851,436, the content of which isincorporated herein by reference, for the synthesis of3-acetoxy-2,2-dimethylpropionic acid.

TABLE VI Substitute cyclopropanecarboxylic acid with following compoundsin DCC/DMAP esterification method: Starting material Examples(structure) Chemical name

3-Acetoxy-2,2-dimethylpropionic acid

3-Pivaloloxy-2,2-dimethylpropionic acid

3-Cyclopropanecarbonyloxy-2,2-dimethylpropionic acid

3-(1-Methyl-cyclopropanecarbonyloxy)-2,2-dimethylpropionic acid

3-(2-Methyl-cyclopropanecarbonyloxy)-2,2-dimethylpropionic acid

3-(2,2-Dimethyl-cyclopropanecarbonyloxy)-2,2-dimethylpropionic acid

3-(3-Tetrahydrofurancarbonyloxy)-2,2-dimethylpropionic acid 64

3-(1-Methyl-3-tetrahydrofurancarbonyloxy)-2,2-dimethylpropionic acid

TABLE VII 3-alkoxy-2.2-dimethylpropionic acids and 3-alkoxyalkyl-2.2-dimethylpropionic acids were prepared by the method described in J. Org.Chem. 38, 2349 (1975). Substitute cyclopropanecarboxylic acid withfollowing compounds in the DCC/DMAP esterificaton method (Example 5c):Starting material (structure) Chemical name

3-Methoxy-2,2-dimethylpropionic acid

3-propoxy-2.2-dimethylpropionic acid

3-isopropoxy-2.2-dimethylpropionic acid

3-Cyclopropylmethoxy-2,2-dimethylpropionic acid

3-(2-Methoxy-ethoxy)-2,2-dimethylpropionic acid

3-Ethoxymethoxy-2,2-dimethylpropionicacid

TABLE VIII 3-N-substituted-2.2-dimethylpropionic acids are prepared bythe method described in U.S. Pat. No. 5,475,013 to Talley et al., thecontent of which is incorporated by reference. Susbstitute cyclo-propanecarboxylic acid with the following compounds in the DCC/DMAPesterification method (Example 5c): Starting material (structure)Chemical name

3-Amino-2,2-dimethylpropionic acid

3-Dimethylamino-2,2-dimethylpropionicacid

2,2-Dimethyl-3-piperidin-1-yl-propionicacid

2,2-Dimethyl-3-(4-oxo-piperidin-1-yl)propionic acid

2,2-Dimethyl-3-thiomorpholin-4-yl-propionic acid

2,2-Dimethyl-3-(4-methyl-piperazin-1-yl)-propionic acid

3-Imidazol-1-yl-2,2-dimethyl-propionicacid

TABLE IX 3-S-substitted-2.2-dimethylpropionic acids are prepared by themethod described in U.S. Pat. No. 5,475,013. Substitutecyclopropanecarboxylic acid with following compounds in the DCC/DMAPesterification method (Example 5c): Starting material (structure)Chemical name

2,2-Dimethyl-3-methylsulfanylpropionicacid

3-Methanesulfinyl-2,2-dimethylpropionicacid

2,2-Dimethyl-3-phenylsulfanylpropionicacid

3-Benzenesulfonyl-2,2-dimethylpropionicacid

TABLE X 3-Substitted-2.2-dimethylpropionic acids are prepared by themethod described in U.S. Pat. No. 5,475,013. Substitutecyclopropanecarboxylic acid with following compounds in the DCC/DMAPesterification method (Example 5c): Starting material (structure)Chemical name

2,2-Dimethyl-3-phenylpropionic acid

2,2-Dimethyl-3-pyridin-4-yl-propionicacid

TABLE XI Various NSAID (nonsteroidal anti-inflammatory drugs containingcarboxylic acid group) are commercially available. Substitute cyclo-propanecarboxylic acid with following compounds in the DCC/DMAPesterification method: Starting material (structure) Chemical name

Ketorolac or 5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid

Flurbibrofen or 2-(2-Fluoro-biphenyl-4-yl)propionic acid

Ibuprofen or 2-(4-Isobutyl-phenyl)propionic acid

Naproxen or 2-(5-Methoxy-naphthalen-2-yl)propionic acid

Aspirin

TABLE XII Various carboxylic acids are commercially available.Substitute cyclopropanecarboxylic acid with the following compounds inDCC/DMAP esterification method: Starting material (structure) Chemicalname

Cyclopent-3-enecarboxylic acid

But-3-enoic acid

Tetrahydro-furan-2-carboxylic acid

Tetrahydro-thiophene-2-carboxylic acid

Tetrahydro-thiophene-3-carboxylic acid

2-Oxo-thiazolidine-4-carboxylic acid

2-Oxo-oxazolidine-4-carboxylic acid

2-Oxo-imidazolidine-4-carboxylic acid

2-Oxo-[1,3]dioxolane-4-carboxylic acid

1-Methyl-pyrrolidine-3-carboxylic acid

1-methyl-pyrrolidine-2-carboxylic acid

Tetrahydro-pyran-4-carboxylic acid

Tetrahydro-thiopyran-4-carboxylic acid

1-Methyl-piperidine-4-carboxylic acid

3-hydroxy-2-methylpropionic acid

3-amino-2-methylpropionic acid

3-mercapto-2-methylpropionic acid

3-methoxy-2-methylpropionate (synthesis:U.S. Pat. No. 4,617,154, thecontent ofwhich is incorporated herein by reference)

1. A composition comprising a pharmaceutically acceptable carrier ordiluent and a compound having the formula:

where R₁ and R₂ are, independently, H or C₁ to C₃ alkyl; R₃ and R₄ are,independently, C₁ to C₃ alkyl; and where R₁ and R₂, taken together, orR₃ and R₄, taken together, or both may be cycloalkyl; R₅ is H, OH, or C₁to C₆ alkyl; R₆ is C₁ to C₆ alkyl, alkenyl, alkynyl, or substitutedalkyl or alkenyl; R₇ is C₁ to C₆ alkyl, alkenyl, alkynyl, or substitutedalkyl or alkenyl; or where R₆ and R₇ form a cycloalkyl having from 3 to7 atoms in the ring.
 2. The composition of claim 1 wherein thesubstituted alkyl or alkenyl has at least one alkoxy, alkylthio,alkylamino, dialkylamino, aryloxy, arylamino, benzyloxy, benzylamino orheterocyclic or YCO-Z where Y is O, N, or S and Z is alkyl, cycloalkylor heterocyclic or aryl substituent.
 3. The composition of claim 1wherein R₆ and R₇, taken together, are cyclopropyl.
 4. The compositionof claim 1 wherein each of R₁ through R₄ is C₁ to C₃ alkyl.
 5. Thecomposition of claim 1 wherein each of R₁ through R₄ is methyl.
 6. Thecomposition of claim 1 wherein R₆ is C₁ to C₆ alkyl substituted with atleast one C₁ to C₆ alkoxy or benzyloxy group.
 7. The composition ofclaim 1 wherein each of R₁ through R₄ is methyl, R₅ is H or methyl, R₆is methyl substituted with benzyloxy or C₁ to C₆ alkoxy, and R₇ ismethyl, or where R₆ and R₇ form a cyclopropyl group.
 8. The compositionof claim 1 wherein each of R₁ through R₄ is methyl, R₅ is methyl, R₆ isethoxy methyl, and R₇ is methyl.
 9. The composition of claim 1 whereineach of R₁ through R₄ is methyl, R₅ is methyl, R₆ is benzyloxy methyl,and R₇ is methyl.
 10. The composition of claim 1 wherein each of R₁through R₄ is methyl, R₅ is methyl, R₆ is hydroxymethyl, and R₇ ismethyl.
 11. The composition of claim 1 wherein each of R₁ through R₄ ismethyl, R₅ is H and R₆, and R₇ form a cyclopropyl ring.
 12. Thecomposition of claim 1 adapted for pharmaceutical use as an eye drop.13. The composition of claim 1 further comprising a reducing agent. 14.The composition of claim 13 wherein the reducing agent is a sulfhydrylcompound.
 15. The composition of claim 1 further comprisingmercaptopropionyl glycine, N-acetyl cysteine, β-mercaptoethylamine, orglutathione.
 16. An ophthalmic composition comprising an ophthalmicallyacceptable carrier or diluent and a compound of claim 1 having anN-hydroxy piperidine portion bound to a solubility modifying portion,the compound having a solubility in water at 25° C. of at least about0.25% by weight and a water n-octanol partition coefficient at 25° C. ofat least about
 5. 17. The composition of claim 16 wherein the N-hydroxypiperidine portion is cleaved from the compound under conditions foundin the eye.
 18. The composition of claim 16 wherein the N-hydroxypiperidine portion is cleaved from the compound under conditions foundin the lens of the eye.
 19. The composition of claim 16 wherein theN-hydroxy piperidine portion is cleaved enzymatically.
 20. Thecomposition of claim 16 wherein the N-hydroxy piperidine portion is1,4-dihydroxy-2,2,6,6-tetramethylpiperidyl.
 21. The composition of claim16 further comprising a reducing agent.
 22. The composition of claim 21wherein the reducing agent is a sulfhydryl compound.
 23. The compositionof claim 16 further comprising mercaptopropionyl glycine, N-acetylcysteine, β-mercaptoethylamine, or glutathione.